The objectives of the proposed studies are to compare and contrast thrombin and properdin factor D. Preliminary studies have demonstrated factor D activity by alpha, beta/gamma and autocatalyzed thrombin. Cross reaction between thrombins and partially purified factor D has been shown with antisera to human prothrombin and to guinea pig factor D. Thrombin and factor D will be purified from human plasma, antisera will be produced in rabbits, and the antigenic composition of the proteins will be evaluated. The IgG fractions of antisera to prothrombin and thrombin will be insolubilized on Sepharose 6B and used to deplete normal plasma, which will then be tested for depletion of factor D activity. We will search for factor D and prothrombin genetic polymorphism by immunofixation electrophoresis to see if sera with factor D variants also have prothrombin variants. Generation of factor D activity from prothrombin will be studied both in vitro and in vivo. Thrombin will be incubated with antithrombin III under varying conditions and reaction mixtures evaluated for factor D activity. Radiolabeled prothrombin will be injected in monkeys and plasma will be collected at intervals after injection. This plasma will be evaluated by isoelectric focusing and SDS-PAGE for labeled protein with the physicochemical characteristics of factor D. Thrombin will be evaluated for its ability to replace factor D in two other assays: the ability to cleave factor B in the presence of cobra venom factor and the ability to replace factor D in the formation of a cellular intermediate containing C3b and factor B on its surface. Each of the three assays will be used to evaluate other coagulation proteins for alternative pathway activation. Hydrolysis of synthetic substrates by factor D, thrombin, factor Xa, and C1s will be compared. Inhibition of esterolytic and hemolytic activity of factor D by DIFP, chloromethyl ketones and benzamidines will be evaluated. The final goal of the proposed studies is to determine the amino acid sequence of factor D to establish its degree of homology with thrombin.